Method and apparatus for treating a biological sample with a liquid reagent

ABSTRACT

A method and apparatus for thin film fluid processing of biological samples without rinsing between treatments is provided. An apparatus having a treatment zone for treating a biological sample with a liquid reagent, comprising first and second substrates having facing surfaces defining a space therebetween in which the biological sample may be treated with the liquid reagent, wherein the first substrate comprises a relatively fluid impermeable element while the second substrate comprises a relatively flexible gas permeable element.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/532,063, filed Dec. 23, 2003, and copending U.S. Ser. No.11/016,407 filed Dec. 17, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus useful in the fluidtreatment of surfaces. It has further utility in the minimization offluid consumables by spreading of a treatment fluid into a thin filmspecific to a designated treatment zone. The invention has particularutility in connection with the fluid treatment of flat substrates andmore specifically staining of biological tissue samples on glass slidesand will be described in connection with such utility, although otherutilities are contemplated.

2. Description of Related Art

The analysis of biological tissue samples is a valuable diagnostic toolused by the pathologist to diagnose many illnesses and by the medicalresearcher to obtain information about a cell structure.

In order to obtain information from a biological tissue sample itusually is necessary to perform a number of preliminary operations toprepare the sample for analysis. While there are many variations of theprocedures to prepare tissue samples for testing, these variations maybe considered refinements to adapt the process for individual tissues orbecause a particular technique is better suited to identify a specificchemical substance or enzyme within the tissue sample. However the basicpreparation techniques essentially are the same. Biological tissuesamples may derive from solid tissue such as from a tissue biopsy or mayderive from liquid based preparations of cellular suspensions such asfrom a smear (e.g., PAP smear).

Typically such procedures may include the processing of the tissue byfixation, dehydration, infiltration and embedding; mounting of thetissue on a glass slide and then staining the sample; labeling of thetissue through the detection of various constituents; grid analysis oftissue sections, e.g., by an electron microscope, or the growing ofsample cells in culture dishes.

Depending on the analysis or testing to be done, a sample may have toundergo a number of preliminary steps or treatments or procedures beforeit is ready to be analyzed for its informational content. Typically theprocedures are complex and time consuming, involving several tightlysequenced steps often utilizing expensive and toxic materials.

For example, a typical tissue sample may undergo an optical microscopicexamination so that the relationship of various cells to each other maybe determined or abnormalities may be uncovered. Thus, the tissue samplemust be an extremely thin strip of tissue so that light may betransmitted therethrough. The average thickness of the tissue sample orslice (often referred to as sections) is in the order of 2 to 10micrometers (1 micrometer= 1/1000th of a millimeter). Typically, atissue sample is either frozen or fixed in a material (a fixative) whichnot only preserves the cellular structure but also stops any furtherenzymic action which could result in the purification or autolysis ofthe tissue.

After fixation, the tissue sample is then dehydrated by the removal ofwater from the sample through the use of increasing strengths ofalcohol. The alcohol then is replaced by a chemical which mixes with waxor some other plastic substance impregnant which can permeate the tissuesample and give it a consistency suitable for the preparation of thinsections without disintegration or splitting.

A microtome is then utilized to cut thin slices from the tissue sample.The slices may be on the order of 5 to 6 micrometers thick while thediameter may be on the order of 5000 to 20000 microns long. The cut thinsections are floated on water to spread or flatten the section. Thesection is then disposed on a glass slide usually measuring about 8 by2.5 centimeters (1×3 inches).

The wax or other impregnant is then removed by exposing the sample to asolvent, the solvent removed by alcohol, and the alcohol removed bydecreasing the alcoholic concentrations until eventually the tissue isonce more infiltrated by water. The infiltration of the sample by waterpermits the staining of the cell constituents by water soluble dyes.

Prior to the development of automated procedures for the preparation oftissue samples, it often took from two to ten days before the tissuecould be examined under a microscope. In more recent years automatedprocesses have been developed utilizing apparatus to transfer the samplefrom one fluid to another at defined intervals, and as a result thepreparation time has been significantly reduced to 12 to 36 hours.

The foregoing discussion of the prior art derives largely from U.S. Pat.No. 5,595,707 to Bernstein et al. which describes an automated systemfor performing a plurality of independent analysis proceduressimultaneously comprising a robotic arm which moves different tissuesamples along a plurality of processing stations arranged along x and ycoordinates wherein the tissue samples are subjected to variousprocesses. See also U.S. Pat. No. 5,595,707 to Copeland et al., whichdescribes an automated slide processing system comprising a reagentcarousel cooperating with a sample support carousel to supply a sequenceof preselected reagents to each of the samples with interposed mixing,incubating and rinsing steps cooperating therewith. Apparatus made inaccordance with U.S. Pat. Nos. 5,675,715 and 5,595,701 and others isavailable commercially from Ventana Medical Systems, Inc. of Tucson,Ariz., and has achieved substantial commercial success and significantlyreduced the time and cost of testing biological samples.

A biological tissue sample is finally viewed by a pathologist in anas-mounted state on a glass slide. Much of the processing of biologicalspecimens, therefore, is adapted to the sequential application andremoval of multiple fluids to an essentially two dimensional treatmentzone on a 1″×3″ glass slide format.

SUMMARY OF THE INVENTION

The present invention provides improvements over the foregoing and otherprior art by permitting a reduction in the amount of fluid volumenecessary to conduct desired biological reactions. Reducing the fluidvolume of reactants results in cost savings of reagents and also resultsin a reduction in the amount of rinse fluids necessary which in turnmeans a reduction in the amount of waste materials that need to bedisposed of. Fluid volume reduction further results in less fluidicmanagement complexity which in turn ultimately permits greater processreliability. It also permits the reduction or elimination of fluid wastedisposal.

The present invention further provides improvements by permitting areduction in processing time to treat biological specimens. Reductionsin fluidic requirements permits rapid treatments and their sequencingwhich in turn permits greater throughput and/or sample turn around time.The present invention further provides that one or more treatmentssurprisingly do not require any rinsing per se, further permitting thereduction of fluidic volume requirements and processing time.

The present invention provides a system, i.e., method and apparatus formanaging micro volumes of fluid. The invention in one aspect providesmethods and apparatus for minimizing fluid volume requirements andprocessing times for performing staining or biological reactions bycreating a staining or reaction chamber formed between a slide and anopposed element such as a hydrophobic element. More particularly, theinvention provides a method and apparatus for spreading a small fluidvolume across a slide surface while providing a regulated passive escapeof trapped gas bubbles and simultaneously avoiding significantevaporative loss.

In a preferred embodiment of the invention a slide is conveyed at anangle to the opposed element to discourage gas bubble entrapment.

The invention is directed to an apparatus having a treatment zone fortreating a biological sample with a liquid reagent, comprising first andsecond substrates having facing surfaces defining a space therebetweenin which said biological sample may be treated with the liquid reagent,wherein the first substrate comprises a relatively fluid impermeableelement while the second substrate comprises a relatively flexible gaspermeable element.

The invention is also directed to a method for treating a biologicalsample with a liquid reagent comprising the steps of providing a sampleand the liquid reagent in the space defined between facing surfaces, andpressing the surfaces together to reduce the space therebetween andexpel gas trapped therebetween.

The invention is further directed to an apparatus for treating abiological sample with a liquid reagent comprising first and secondsubstrates having facing surfaces defining a space therebetween in whichthe biological sample may be treated with the liquid reagent in atreatment zone, wherein the first substrate comprises a relatively fluidimpermeable element while the second substrate comprises a gas permeableelement, the apparatus further including a device for separating thefirst and second substrates downstream of the treatment zone.

The invention is also directed to a method for treating a biologicalsample with a liquid reagent comprising the steps of providing thepreceding apparatus for treating the biological sample, providing thesample in liquid reagent in the space defined between the facingsurfaces, pressing the substrates together to reduce the spacetherebetween and expel gas trapped therebetween and separating saidsubstrates.

BRIEF DESCRIPTION OF THE DRAWINGS

Yet other features of the present invention will be seen from thefollowing detailed description taken in conjunction with theaccompanying drawings wherein:

FIG. 1 compares wetting and non-wetting of a surface by a fluid dropletin accordance with the prior art;

FIGS. 2 a-2 c illustrate fluid droplet behavior of wetting andnon-wetting of fluids placed between two surfaces in accordance with theprior art;

FIG. 3 illustrates vapor lock problems common when a fluid is placedbetween two surfaces in accordance with the prior art;

FIGS. 4 a-4 c illustrate schematically and FIG. 7 illustrates across-sectional view of one preferred embodiment of the presentinvention;

FIGS. 5, 6 a and 6 b are views similar to FIG. 7, and illustratealternative embodiments of the present invention;

FIGS. 8 a and 8 b illustrate manual separation of the slide from asubstrate in accordance with the present invention;

FIGS. 9 a and 9 b illustrate mechanical assisted separation of the slidefrom a substrate in accordance with the present invention; and

FIGS. 10 a and 10 b illustrate a laminated membrane article of thepresent invention;

FIG. 11 illustrates a bench system made in accordance with the presentinvention;

FIG. 12 diagrammatically illustrates a process scheme of one embodimentof the invention; and

FIGS. 13 a and 13 b are views similar to FIGS. 9 a and 9 b ofalternative systems made in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Before considering the present invention in detail, a review of thephenomenon of wetting and spreadability would be useful for a properunderstanding of the present invention.

Spreadability refers to the relationship of a fluid on a surface. Thereis a balance of forces that determine whether or not a fluid has atendency to spread with respect to said surface:

i) fluid-fluid interaction (tension)

ii) fluid-surface interaction

iii) environment (usually air) interaction with fluid and surface

Referring to FIG. 1, consider a fluid droplet 20 resting on a horizontalsurface 22. Absent other forces, the fluid droplet, in an effort tominimize its surface would be drawn into a sphere due to its surfacetension. In other words, when fluid-fluid forces dominate, fluidinteracts more strongly with itself rather than the surface. By way ofexample, and as applied to aqueous (H₂O) based systems, hydrogen bondingin H₂O based fluids is sufficiently strong such that in manycircumstances aqueous fluids will potentially interact fluid-fluidrather than fluid-surface; e.g., when a surface exhibits relatively lowinteraction with the fluid, i.e., the surface is “hydrophobic”, if thefluid is de-ionized H₂O. The fluid will tend to “ball up” on the surfaceand readily can be destabilized, e.g., can be made to roll off of thesurface with modest agitation. However, gravity and interfacial tensionsbetween the liquid droplet and its surroundings usually act against thissurface tension so that the liquid droplet assumes other shapes.Hydrophilic surfaces are those wherein the aqueous-based fluid-surfaceinteraction is significant. When fluid-surface interaction forces arestrong, fluid will preferentially contact the surface and thereby spreadout on the surface.

Contact angle indicates this balance of forces. A contact angle greaterthan 90 degrees indicates relatively weak fluid-surface interactionforces; a contact angle less than 90 degrees indicates relatively strongfluid-surface interaction and spreadable condition. Surface“wettability” is defined as strong fluid-surface interaction withtendency for fluid spreading.

In a spreadable condition, the fluid will tend to spread. This is athermodynamic condition. It may not spread, or spreading may beconstrained or mediated over time due to kinetic limitations. Using anopposing contacting surface, a fluid can be made to spread very rapidlyallowing the thermodynamic state to be satisfied. The contacting surfacemay or may not be spreadable with respect to the fluid. With a fluidbetween two opposing surfaces, only one surface needs to be interactivewith the fluid such that it spreads. Thus, in the case of a surfacewettable by the liquid, the fluid droplet 20 spreads along the surface22. The angle θ formed between the fluid and solid is called thedihedral angle or contact angle. In the case of total wetting θ equals0°. In the process of spreading however, gas pockets may inadvertentlybecome entrained and entrapped within the resulting fluid layer. Thepresent invention, in part, provides a solution for resolving entrainedor entrapped gas pockets.

FIGS. 2 a-2 c and FIG. 3 illustrate what happens when a slide and asubstrate in accordance with the prior art, with wetting and non-wettingfluid-surfaces, are brought together. Referring in particular to FIGS. 2a-2 c, as the surfaces 22 a and 22 b are brought closer together,initially the fluid droplets 20 join to form a larger fluid body 24. Asfluid spreads within the interfacial gap between the surfaces 22 a and22 b, one or more gas pockets 26 may become entrapped as the fluid bodyassumes a thin fluid film shape. If the fluid volume is sufficient,essentially all the space directly between the two opposed surfaces willfill with fluid and assume that shape. Some portions of that shape mayfill before others and connect with other advancing fluid portions. Inthis manner, slower portions may never have a chance to wet because theybecome encircled by advancing portions and thus become “vapor locked”with a pocket of associated gas.

Alternatively, gas pockets may form, post spreading, from dissolvedgases coming out of a saturated solution due to changes in localpressure and/or temperature.

Treatment fluids especially when small in volume need somehow to bereliably administered across a complete treatment area and spatiallymanaged there. For example, for the staining of biological specimensplaced onto a 1″×3″ microscope slide, fluids need to be first placed andthen removed from a rectangularly shaped flat surface. As discussedabove, small fluid volumes tend to remain in droplet formation tominimize thermodynamically driven surface tension forces between liquidsand atmosphere. As a result, fluid spreadability across asurface—especially when fluid volumes may be minimal—can be a realchallenge. For instance, fluids may sit on a surface as droplets ratherthan a continuous thin film. Flooding a surface is one method to resolvethis problem; however, flooding entails the use of a large volume orfluid which is not economical and also requires the management of largereservoirs, waste systems, and complex plumbing. Additional volume alsois needed to fill the rectangular shape of the slide at the corners. Onesimple approach to solving these fluid dynamic challenges is to simplyimmerse the entire substrate or the treatment section of the substrateinto a body of fluid. Early treatment methods as well as someconventional manual methods have exploited this “bucket chemistry”approach. However, the contemporary state of the art has successfullyexploited “fluids on a slide” approach to great effect, especially inregards to automation of fluidic treatments. In this approach, unless alarge flooded volume approach is used (e.g., as in apparatus availablefrom DAKO Cytomation AS, Copenhagen, Denmark), some means ofenchambering is employed to better manage (minimize and control) fluidicvolumes on the slide. Previous prior art methods strive for precisevolume control, and in some cases with volume minimization in mind(hybridization processes because of reagent cost), have employed variousforms of enchambering (e.g., LIQUID COVERSLIP™—Ventana Medical Systems,Inc., Tucson, Ariz.; slide chambers—Cytologix Corporation, Cambridge,Mass.; chamber walls—Agilent Technologies, Inc., Palo Alto, Calif.;chamber walls—Amersham Biosciences, Piscataway, N.J.; TechMate Mcapillary glass slides—Biotek Solutions, Inc., Santa Barbara, Calif.).Conventionally, seals of one sort or another are used to controlon-slide volumes via fixed chamber boundaries. Providing a “liquidtight” chamber further allows pressurization of the fluids as well as insome instances mechanical motion of the walls providing fluid transportto and from the chamber and/or mixing.

However, the need to both contain and specifically place a fluid withrespect to a substrate presents various technical challenges andproblems. Sealing means entail specific and precise coupling of elementsthat are generally encumbering to a process. Two opposing elements witha mediating seal generally require precise alignment and attention toproper maintenance (e.g., cleanliness) of the critical surfaces. Sealsare subject to defect and failure. For example, a seal involving amediating O-ring (or gasket) captured between 2 surfaces may becompromised by a defect in the O-ring, a twist in the O-ring, orcontaminant particles on any of the critical mating surfaces. In anotherexample, a seal involving an adhesive may be compromised by defects inthe adhesive, voids in the adhesive, or contaminant particles. Also,sealing is generally performed directly to the substrate itself. Thesealing quality of incoming substrates with different processes withvarious customers may be a significant variable difficult to manage fora robust seal-dependent process. The present invention addresses theforegoing and other technical problems while also solving the importantissue of entrapped bubbles within the fluid thin film without resort tosealing means while also avoiding significant evaporative loss. In thepresent invention, the chamber “sides” are not physically bounded andthe opposing substrate surfaces may be either fixed or allowed to floatwith respect to one another.

The present invention is used for advantageously performing staining orbiological reactions of biological samples on slides. Referring to FIGS.4 a-4 c, 5, 6 a and 6 b the principle of operation is as follows. Anaqueous based fluid droplet 30 can be spread as a thin layer 32 whenplaced between two surfaces 34, 36. If both surfaces are hydrophilic,surface interactions will contribute to liquid spreading. However, gaspockets 38 (see FIG. 3) may form in the gap between the two surfacesduring fluid spreading or after spreading due to dissolved gases comingout of solution. If both surfaces are non-permeable to gas (e.g.,glass), any trapped or formative gas pockets essentially become fixed inlocation disrupting continuity of the liquid layer (otherwise describedas a condition of “vapor lock”). Liquid tension forces are inhibitedfrom collapsing the gas bubble by the exerted vapor pressure of the gasbubble. In other words, there is no place for the gas to go. Further,viscous forces dominate in thin capillary geometries such that the gasbubbles 38 can be exceedingly difficult to dislodge. Referring inparticular to FIGS. 4 c and 5, the use of a gas permeable material 40 asone of the surfaces in accordance with the present invention providespassages 42 for escape of the gas under the influence of liquid tensionforces. Surface properties are important because if the surface of thegas permeable material 40 strongly interacts with the liquid, liquidwill be absorbed, blocking access to the permeable element, and thebubble remains trapped between the two liquid-coated surfaces asillustrated in FIG. 6 a. On the other hand, if the surface is largelynon-interactive with the liquid, no blocking occurs and the gas bubbleis effectively expelled by the driving force (which is passive) of theliquid tension as illustrated in FIG. 6 b. Favoring this process is lowspreadability of the fluid at this surface; e.g., high surfacehydrophobicity and high surface tension of an aqueous-based liquid.

Thus, the present invention in one aspect is based on the provision of ahydrophobic high gas permeable material 40 for forming a boundarysurface of an open sided reaction chamber to provide pressure relief for“burping” gas pockets from within a thin liquid film. A microporoushydrophobic material such as Gore-Tex® is preferred for its very highgas permeability. High water entry pressure (WEP) materials are furtherpreferred so as to prevent blow-through of liquid. High water entrypressure can be attained by using small pore sized (0.05 to 200 micron)material, or by using a porous hydrophobic material laminated to aliquid-impermeable backing 50 as illustrated in FIG. 7. Aliquid-impermeable backing prevents blow-through of a liquid even if theporous hydrophobic element has large pore sizes (e.g., 200 to 2000microns). It is preferred that the material 40 present a flat smoothsurface for efficient fluid spreading since any surface depressions canresult in localized pooling of fluid impacting spreading efficienciesfor very low volume applications. Smaller pore size hydrophobicmaterials (less than ˜200 microns) provide for a more flat smoothsurface. Moreover, any significant warp in the material may result inlack of fluid coverage. It is also preferred that the material 40 isdimensionally stable, but flexible so that it may be manipulated,meaning that it can be slid over surfaces or rolled around rollers, forexample.

One presently preferred material 40 is “Plumbers Tape”, i.e., Teflon®tape, commonly used on pipe threading to prevent water leakage. PlumbersTape is useful since it meets the following criteria:

-   -   1) It is hydrophobic—comprised of Teflon®        (polytetrafluoroethylene);    -   2) It is microporous—the Teflon® is expanded resulting in        microporosity in the range of 0.05 to 5 microns;    -   3) It has relatively high water entry pressure—it is relatively        dense with very small pores;    -   4) It is flexible and conformable—it can assume the        dimensionality (flatness & smoothness) of a backing element or        surface; and    -   5) “Stiction” is high—it tends to grip smooth hard surfaces if        pressed onto such surfaces.

Since evaporation across a high surface area permeable material may behigh, in order to regulate evaporation, material 40 preferably is backedby a substantially non-permeable material 50. Thus, vapor pressure (andvapor lock relief) between the fluid phase and atmosphere is mediatedlaterally through the permeable material 40 and restricted to thesurface area exposed just at the edges 52 of the article. Preferred as ahydrophobic micro-porous permeable material 50 is 1.5″ wide MilitaryGrade Teflon® Thread Tape available from McMaster-Carr Supply Company,Atlanta, Ga., P/N 6802K66 (“Plumber's Tape”), which provides rapidpressure equilibration, backed by a substantially non-permeable flexiblemembrane or coating. Other suitable porous hydrophobic materials includesiliconized paper and porous polypropylene membrane.

FIGS. 4 a-4 c illustrate in a (general way the manner by which asubstrate 34, a fluid droplet 30, and a glass specimen slide 36 arebrought together in accordance with the present invention. As can beseen, as the glass specimen slide 36 and substrate 34 are brought closertogether the liquid droplet 30 begins to spread thinly and uniformlybetween the opposing surfaces with gas bubbles being effectivelyexpelled at the membrane surface and out through the edges of thearticle.

Microscope glass slide dimensions are nominally 1″×3″ ConventionalSuperfrost™ slides from Erie Scientific Company, Portsmouth, N.H. whichinclude a 0.75″ label region at the end of the slide, leavingapproximately 2.25″×1″ active area. Some microarray slides use largeractive areas and consequently smaller or no label areas. In the presentcase, an active area of 2″×1″—or 5.0×2.5 cm—is assumed. Actual activeareas can be accordingly adjusted upwards or downwards, depending uponapplication.

A 5.0×2.5 cm area equates to 12.5 cm². This was the actual test areaused for stain processing and characterizing volumetric ranges in thefollowing examples. Using less than the total glass slide area allowsfor handling of the slide via gripping the label-end. Using less thanthe total membrane article area allows for handling/gripping of saidarticle.

An applied fluid volume range can be defined wherein practical lower andupper limits are described. This can be defined in terms of per slidebasis; however, it is clearer to define in terms of per Cm² basis sincethe active area may vary as explained above.

At the lower fluid volume end, 0.0007 ml per cm² with a fluid layerthickness of 7 μm was about as thin as could be fully spread over theentire contact area for a given aqueous-based fluid, glass slide, andgiven contacting membrane. Spreading did not occur spontaneously butrequired some work; i.e., the two surfaces required some pressure P tofacilitate spreading. Furthermore, it was advantageous to move oroscillate one surface with respect to the other to help “drive” thefluid throughout the gap between the surfaces. At this lower limit,viscous forces of the fluid are significant such that the “flow” of thefluid is retarded. Thus, at the lower volume end additional stepssometimes may be required to move fluid throughout the gap so as toattain thermodynamic equilibrium of a fully wetted contact area.Additionally, at the lower volume end entrapped gas pockets did not seemto readily “burp”. Slow burping may very well be associated with highviscous forces retarding the movement/collapse of the entrappedgas-liquid boundary.

Fluid volumes of 0.002 to 0.0055 ml per cm² (20-55 microns thick fluidlayer) were found to be a middle range where volumes could be minimizedwithout incurring severe viscosity issues. Both spreading and burpinggenerally occur spontaneously within this range, the greater the volume,the more spontaneous.

Fluid volumes greater than 0.0055 ml per cm² up to approximately 0.036ml per cm² can also be useful (55 to 360 microns thick layer). Burpingand spreading proceed readily. As volumes increase, however, fluids atthe boundaries may have a greater tendency to be inadvertently squeezedout of the contact area and pinched off from the main fluid body.Droplets may then end up at the edge or outer surfaces of the slideand/or membrane in an uncontrolled manner. Inadvertent droplet formationthus becomes more sensitive to contacting pressure and its control asvolumes increase. Inadvertent droplets may not necessarily be a problemper se, but require some attention as to management.

Furthermore, higher volumes are akin to “floating” one of the substratesover the other—viscous forces no longer appear so dominant. One effectis that the fluid layer can act as a “fluid bearing”; e.g., the topsubstrate can readily roll off of the bottom substrate with a slighttilt of the apparatus. This property can be exploited for separation ofthe two substrates post treatment. It also would require fixturing thetwo substrates to prevent uncontrolled separation during treatment.Conversely, with low volumes, the apparatus can be severely tiltedwithout effecting separation of the two substrates due to the highviscous forces at work.

Thus, any three of these defined volume ranges may be useful, not justthe lowest volume regimes. Volume requirements are traded againstcertain characteristic features that offer specific designpossibilities. Higher reagent volumes than currently used may thus bepreferred depending upon the preferred design space.

Actual range values may vary according to the specific fluids andsurfaces used. In the previous example, values reflected DI water withSuperfrost™ glass and Teflon® Plumbers Tape Military Grade, While actualvalues might vary accordingly, low, middle, and high volume ranges arethus characterized providing specific design spaces for consideration.In the case of a slide, the targeted “treatment zone” is provided by acontact area. The contact area is determined by the opposition of firstand second substrates (substrate and slide surfaces) when broughttogether involving an intermediary fluid. If one of the two surfacesterminates at a particular boundary, the fluid boundary essentiallyco-terminates at this boundary as well—in effect, we have a “controllingsurface” defining the shape of the thin fluid body. Conversely, thealternate surface may extend without effect on the fluid shape since itis non-controlling. Either surface can be controlling. This isillustrated in FIGS. 10 a and 10 b. This is important because theboundaries of the thin fluid layer can be thus managed while thephysical dimensions of one of the surfaces (at a time, per boundary) canbe relaxed allowing for, e.g., non-treatment of the label area of theglass slide and over-sizing of the substrate, e.g., for handlingpurposes, as will be discussed below.

The ability of the present invention to eliminate gas bubbles and spreada relatively small liquid volume across a relatively large surface areawhile advantageous, also may present a technical challenge in subsequentseparation of the specimen slide from the substrate due to significantliquid adhesive forces between the specimen slide and the substrateespecially at lower volumes. Thus, the present invention, in anotheraspect, provides a system for facilitating this separation. FIGS. 7, 8 aand 8 b illustrate separation of the glass specimen slide 36 from thesubstrate 34 in a manner that deals with the significant liquid tensionthat results between the specimen slide 36 and the substrate 34, by“peeling” of the flexible substrate 34 away from the specimen slide 36.Various manual and/or mechanical means for lying down and peeling backthe flexible substrate 34 onto and from the specimen slide 36 areenvisioned. FIGS. 8 a and 8 b illustrate manual separation and FIGS. 9 aand 9 b illustrate an embodiment wherein belt drives 60, 62 actuate bothpeeling and separation of the flexible substrate 34 from the specimenslide 36 while simultaneously damping off and wicking away spent fluidvolumes on absorbent belts 64, 66 carried on the belt drives 60, 62.

The first substrate comprises a relatively fluid-impermeable element.Typically this substrate is glass, plastic or metal that holds thebiological substrate sample. The second substrate comprises a relativelyflexible gas permeable element, and includes the Gortex® membranesmentioned above.

The invention will be further illustrated by the following exampleswhich are intended to illustrate the invention and not limit it in anyway.

Materials List

Substrate: Paraffin-embedded tonsil sections mounted on precleanedSuperfrost@ Plus microscope slides, 25×75×1 mm (Erie ScientificCompany), were air dried and stored in a slide box for several daysprior to treatments.

Fluidic device-contacting membrane element: Metricel® 47 mm disks 0.1 umporosity polypropylene, Pall Corporation, East Hills, N.Y. P/N M5P4047.

Fluidic device-backing element: Transparent single side adhesivesilicone sheeting 0.020″ thickness, McMaster-Carr Supply Company P/NR700549PK.

Fluidic device construct: From silicone sheeting were cut out a˜2.7×˜6.5 cm rectangular section. The adhesive backing material wasremoved from all but the last 2 cm of the sections—this end serves as ahandle for the final device. Over the exposed adhesive, a 47 mm disk wasoriented and adhered. The laminate was then trimmed to remove excessoverhang materials. Because of the curvature of the disk, the siliconesheeting was trimmed with a slight curving of the rectangular corners tomaximize available contacting area for treatment. (See FIG. 10 a). Finalarea of the membrane surface is ˜2.7×4.4 cm. The same deviceconstruction was used through all aqueous steps involved throughimmunohistochemical staining (IHC).

Solvent fluidic device: Since non-polar solvents wet out the abovedescribed device, another material was used in treating the samplethrough the depar and dehydration operations. ˜7 cm length ofTissue-Tek® SCA™ coverslipping film (Sakura Finetek USA, Torrance,Calif., P/N 4770) was used. One side contains adhesive—the non-adhesiveside was used to spread solvents for treating the sample. The samematerial was used through all non-polar steps in depar and dehydration.

Coverslipping: A 5.2 cm length of the same coverslipping material wasused to coverslip the sample in the final operation.

Wicking Pads: Two were used, one for absorbing aqueous solutions, onefor non-polar solvent collection. Gel Blot paper P/N GB003 fromSchleicher & Schuell Bioscience, Inc., Keene, N.H., was used, cut to 3×6cm sections. A small piece of Scotch® Tape (3M Company, St. Paul, Minn.)was taped to the last 0.5 cm ends and then adhered back onto itself toact as a handle. For the aqueous pad: it was soaked in Reaction BufferConcentrate (10×) diluted to 1:10 in deionized water (Ventana MedicalSystems, Inc., Cat #950-300). Upon soaking, excess was squeezed out bymild compression of the wetted pad. The objective was to have a moistabsorbent pad for effective wicking. The majority of the originallyabsorbed solution was squeezed out such that the pad was “damp” ratherthan “soaked”.

Staging area: A 5×7.5×9.5 cm aluminum block 80 was used as a stage forplacement of the slide and articles during treatment [FIG. 11].

Tools: a VWR brand pipette 0.010-0.100 mL (VWR International, Inc., WestChester, Pa.) range was used for collecting and dispensing precisevolumes of reagent.

Reagents: Confirm™ Anti-CD34 (Clone QBEnd/10) Ventana Medical Systems,Inc. cat# 790-2927 was used as the primary antibody designed to stainvascular endothelial cells. The secondary antibody was UniversalSecondary Antibody Ventana Medical Systems, Inc., P/N 760-4205 VentanaMedical Systems, Inc. The other reagents were from DAB MAP™ Kit cat#760-124 Ventana Medical Systems, Inc. The Blocker D reagent was not usedin this example in order to demonstrate just how bad the backgroundmight get as a result of processing without the benefit of a blocker.Each reagent (˜0.100 ml) was transferred into 0.2 ml microfuge tubes foruse.

Method

Staging: Referring to FIGS. 11 and 12, set up flat top aluminum block orstage 80 on end on lab bench. There is an orientation option—either theglass slide may be placed first onto the block and the membrane articlewith fluid sandwiched in between placed on top, or, the membrane articlemay be placed down first and the glass slide placed on top. In terms oftreatment, it has been found to be immaterial which orientation isselected. Since a fluid drop is dispensed as an intermediary step, itwas found generally for manual application that it was more convenientto first place the membrane down, followed by the fluid dispense,followed by opposition (sandwiching) of the glass slide on top. Thisalso had the added advantage in that one could visually observe fluidicbehavior through the backside of the transparent glass from a top view.With low volume applications, over expression of fluid at the boundariesis of little to no concern since there is so little excess and since thefluid behaves in a highly viscous manner facilitating fluid placementcontrol. At higher volume applications, however, fluid may tend toover-express at the boundaries and excess management may become aconcern. With over-expression, care should be taken with respect tostage design such that no wicking or contacting surfaces from the stageare near any fluid boundaries (e.g., use a stage slightly smaller insize than the mounted substrate). Such a design provides for a robustprocessing area where a wide range of volumes may be exercised. In thepresent case, the membrane article was placed flat and more or lesscentral onto the block such that when the slide was then placed down,the label end 82 of the slide overhung the edge of the stage. (See FIG.11). This allowed for ready access and handling of the slide. Picking upthe slide also picked up the membrane article “adhered” via the fluid tothe slide. This “sandwich” could be safely manipulated in space—turnedaround and upside down—without disruption of the elements, fluid, ortheir relative dispositions.

Contacting Area: The membrane surface area is ˜2.7×4.4 cm. Thedesignated glass slide treatment area is 2.5×˜5.0 cm. The glass slidewidth (2.5 cm) dictates the width of the contacting area whereas themembrane length (4.4 cm) dictates the length of the contacting area. Inother words, the membrane overhangs the glass in the width dimensionwhile the glass overhangs the membrane in the length dimension. (SeeFIGS. 10 a and 10 b). The resulting contact area is thus 2.5×4.4=11 cm².The tissue section was placed well within the treatment area. 0.020 mlfluid volumes were used for all fluid applications, since this waspreviously determined to be sufficient for both coverage and burpingpurposes. This resulted in a 0.020/11 or 0.0018 ml per cm² “fluidicoperational” value. This is at the low volumetric end or viscous regime.High viscosity was clearly observed, but there was not a single incidentof failure or retardation of burping observed at any step.

IHC treatment—dispenses: In order to perform a properimmunohistochemical stain, a specific series of reagents must be appliedfor specified time exposures. Concentrations are established by the kitmanufacturer. It was decided to use all the reagents in the fullconcentrated form without dilution. This provided for acceleration oftreatment such that a standard 2 minute exposure time could be used forevery step. Different exposure times were not tested nor optimizedbeyond this setting. 0.020 ml fluid volumes were applied to the slidefor each dispense. It was also decided to eliminate all rinse dispenseswith the exception of one applied after the SA-HRP treatment. Anunexpected finding was that rinse applications were not necessary at anyof the reagent steps with the one exception—as long as the bulk of thereagent volumes could be removed by other means. For the singular rinsestep, Reaction Buffer concentrated (10×) diluted 1:10 was used (0.020ml). Placement of the dispensed volume was not critical as the act ofsandwiching spreads the fluid evenly between the opposing surfaces. Thisis significant in that with automation, instrumentation design may berelaxed on this point. Dispenses were generally placed somewherecentrally onto the fluidic article. The first dispense involvedInhibitor D. It was found that 0.020 ml barely covered the full contactarea. Subsequent applications of the other reagents exhibited nodifficulty with coverage, on the other hand. It is possible that thefirst use of a virgin membrane surface is sub-optimal and not preferred.

Thus, a pre-conditioning where the membrane is exposed to proteinabsorption may enhance spreadability in subsequent steps.

Treatment Sequence:

1) Inhibitor, 2) Anti-CD34, 3) Universal Secondary Antibody, 4) SA-HRP,5) Reaction Buffer Rinse, 6) DAB+DAB H2O2, 7) Copper.

In step 6, two reagents were applied together. In the present case,0.020 ml of DAB was pre-mixed with 0.020 ml of DAB H2O2 just prior toslide dispense. Only 0.020 ml of the mix was applied.

IHC treatment—fluid removal: After each treatment of 2 minute, thesandwich was picked tip into the air by the glass slide label end, thehandle end of the fluidic article was grabbed, and the membrane peeledaway from the slide surface. The fluidic device was then returned to thealuminum block or stage, the moistened blotting paper was placed on top,and then the glass slide was placed on top of the blotting paper. Slightpressure was applied to the top of this new sandwich such that excessfluids at both the fluidic and slide surfaces could be readily absorbedinto both blotting paper surfaces simultaneously. Total time to performsuch a wicking operation was between 10 and 20 seconds, largelydepending upon manual dexterity. Actual wicking time was probably—lessthan −5 seconds.

Solvent deparaffinization treatment: A specific series of solventexposures were applied in order to effectively remove paraffin whilereturning tissue back to an aqueous state. The treatment operations wereessentially the same as those described for the IHC operations with afew notable differences. The glass slide was placed face up onto thestage with the fluidic article placed on top sandwiching thefluids—upside down with respect to aqueous processing. The fluidicarticle (Sakura coverslip plastic strip) is quite thin, so it was easierto prevent fluids from exceeding their boundaries with the glass slidesitting with its wetted surface 1 mm above the stage surface. Further,the plastic is transparent so visualization was sufficient. The mostsignificant difference is that the fluidic device in this instance isnon-burpable. Sliding the plastic strip back and forth over the “fluidbearing” provided adequate exposure of the tissue to the solvents inspite of entrapped gas pockets. This method is an important alternativeand unique method for assuring full coverage treatment, one thatmitigates the effect of entrapped gas pockets.

Since fluids are directly applied to the treatment area, it is importantto immediately sandwich the fluidic device with the slide forhomogeneous treatment. Solvents in all cases were applied for only ˜10seconds per application. Wicking was performed the same way as in theIHC aqueous case, except that a separate wicking pad (dry) was used forcollecting excess non-polar solvents. 0.020 ml volumes were used perapplication.

Treatment Sequence:

1) Xylene (repeated 3×), 2) 100% ETOH (repeated 2×), 3) Reaction Buffer(just once)

At the end of treatment (while in step 3), tissue was parked andtherefore “soaked” in Reaction Buffer for several minutes as the IHCprocess was being set up.

Dehydration Treatment: Essentially the same operations as thedeparaffinization were applied, just in reverse. The same solventwicking pad was used.

Treatment Sequence:

1) 100% ETOH (repeated 3×), 2) Xylene (one time), 3) Xylene+Sakuracoverglass.

It was observed that 0.020 ml was an insufficient volume for thecoverglass step 3. An additional 0.020 ml was added around the finishedcoverglass to displace the air and supplement the originally appliedvolume.

There was no intermediary step between final treatment of Copper reagentwicking during the IHC operation and dehydration—the sample wentstraight from IHC to solvent applications.

Final Results and Advantages;

Total process time from wax to coverglass=20 minutes. This is very fastwhere analogous process of using conventional prior art techniques wouldtake ˜90 minutes just for the depar+IHC operations alone.

Total fluids consumption: 0.160 ml aqueous; 0.120 Xylene; 0.100 ETOH.This is ˜1000× reduction compared to conventional processes.

Total fluids stream waste: none; only 2 moist wicking membranes.

80% reduction in expensive reagent volumes (0.020 ml treatment comparedto 0.100 ml on the BenchMark, per reagent).

Elimination of all but one rinsing step. More economical; faster.

Elimination of all mixing requirements; no mixing overhead

Both stain and background are acceptable.

The elimination of holding steps between depar, IHC, dehydration, andcoverslipping operations—a continuous process from deparaffinizationthrough coverglassing.

The ability to operate with extremely small quantities of liquid reagentprovides several features and advantages over the prior art. For one,stains and reagents may be used in concentrated, undiluted form. As aresult, liquid volume may be significantly reduced, and wash or rinsevolume also may be significantly reduced. Also, depending on the natureof the reagent or stain and subsequent operations, it may be possible toeliminate one or more wash or rinse steps. This latter feature andadvantage to the present invention is quite unexpected and contrary tocurrent practices of the prior art in which heretofore it has beenaccepted that rinsing between applications of subsequent reagents isinherent to the processes involved in the proper (i.e., clean) stainingof slides. By employing a hydrophobic flexible substrate 32 inaccordance with the present invention, it is possible to displace spentreagent or stain by other means than rinsing. In other words, it hasbeen discovered that reagent displacement alone sometimes may besufficient for processing slides through staining. Therefore, inaccordance with another aspect of the present invention alternativedisplacement means can be provided which eliminates the need for anyrinsing operations. For example, spent stain or reagent may be removedby wicking using a blotting paper or cloth such as Gel Blot Paper (GB002or GB003, available from Schleicher & Schuell BioScience, Inc.) (seeFIGS. 9 a-9 b), and the slides further treated without any rinsing.Alternatively, the spent stain or reagent may be removed by air knifingor spinning (see FIG. 13 b). This has the advantage of resulting in areduction in total fluid volumes of ˜1000× compared to presentlycommercially available systems. No liquid waste stream need begenerated; the only waste involved are small aqueous-moist andnon-polar-moist wicking pads derived from reagent collection. In otherwords, the present invention permits reduction in liquid consumables andwaste by elimination of rinsing or washing between one or more stainingor reagent steps.

The ability to satisfactorily conduct one or more reagent steps withoutintervening wash or rinse is unexpected and contrary to the currentlyaccepted legacy coming from the old “bucket chemistry” days, ingraineddogma, and general heightened concern surrounding background,carry-over, and the ideal of chemical isolation. With pressure towardsvolume reduction, process acceleration, and improved instrumentreliability, processes such as rinsing are coming under greaterscrutiny. The present invention provides an optimal method forauto-staining slides that eliminates many of the disadvantages inherentin prior art systems.

Referring to FIGS. 13 a and 13 b there are illustrated yet otheralternative embodiments of the present invention which permit control ofslide entry angle and motion to discourage bubble entrapment andmitigation of unintended bubble entrapment. In FIG. 13 a the systemincludes a base station 80 upon which a replaceable surface element 84is fitted. Element 84 may comprise a fixed article, such as an injectionmolded plastic plate, that can be periodically replaced with a newpiece. In this manner, a well-controlled fluidic surface quality may bemanaged. Alternatively, as shown in FIG. 13( b) the surface element maycomprise a membrane sheet 86 threaded and held tautly between a feedroller 88 and a take-up roller 90 which permits fresh membrane to beadvanced, as necessary, and thus provide fresh surface assuringwell-controlled fluidic surface quality. A dispenser 92 dispenses afluid drop 94 of known volume onto the right entry side of element 84 or86 as the case may be. A slide 96 is conveyed (details of the conveyancemeans not shown for the sake of clarity) initially at an angle so as tocontact the fluid drop 94 and then oriented in parallel with the surfaceplane of element 84 or 86 as the case may be as it is further conveyedto the left. Conveyance of slide 96 may be independent of conveyance ofelement 84 or 86, as the case may be. Keeping conveyance independent ofthe relative motion of slide 96 with respect to element 84 or 86 as thecase may be in combination with angled contacting of the slide withdroplet 94 and motion to parallel orientation with element 84 or 86 asthe case may be ensures essentially bubble-free fluid spreading andcoverage within the resulting capillary gap. The capillary gap isdefined as any continuous opposition of slide 96 with element 84 or 86as the case may be in which fluid can fill. Slide 96 is conveyed acrossthe surface of the device reaching the far side at left. The speed ofconveyance controls the time of slide 96 surface exposure (incubation)to capillary gap fluid. Throughput may be increased by increasing thelength of the base 80 and/or by incorporating heating means 98 or 106into the base 80. With heating the speed of conveyance may be increasedin order to maintain equivalent incubation effect thereby increasing thecapacity to process more slides. As slide 96 is further conveyed, it isincrementally removed from the device. Most fluid tends to remain withinthe diminishing remaining capillary gap. Upon conveyance beyond theboundary of element 84 or 86 as the case may be, the majority of thefluid spills down the side of device as fluid droplets 100 leavingtreated surface of the slide 96 largely free of excessive residualfluid. Slide 96 may be further conveyed to another location and treatedto remove additional surface fluid, if needed, for example by an airknife 102 or absorbent porous membrane surface element 104 treatment.Additional like base stations and dispensers, etc., may be used inseries in order to affect a series of chemical surface treatments in thesame manner.

Also, a heater element 98 or 106 may be incorporated into element 84 orthe entire system may be placed within a chamber with controlled heatingcapabilities.

It is thus seen the present invention provides significant methods andsystems for staining or incubating micro volumes on substrate surfaces.Fluid volumes on the scale of 20-50 ul can be spread as thin layers overlarge surface glass slide areas (12.5 cm² area>1.6-4.0 ul/cm²). Thefluid on the slide may be maintained in a non-sealed manner such that aspecified treatment area is contacted for appropriate fluidic exposure.Maintenance of a continuous thin aqueous layers without disruption is arecognized challenge. The present invention provides a system forspreading a small liquid volume across a large surface area whileproviding a regulated passive escape of trapped or formative gas pocketswhile avoiding significant evaporative loss.

The invention has other advantages. With direct concentrated reagentapplication, processes may be accelerated and the requirement of mixingreagent with a diluent eliminated. Application of concentrated reagentsalso means that liquid volume dispense precision may be relaxed, sinceconcentration, not volume, becomes the controlling factor.

The invention is useful in providing well-controlled serial incubationsof specific chemistries. Elimination/reduction of heavy rinsing incombination with small reagent volume applications suggests newarchitectural opportunities, such as miniaturization, systemintegration, and elimination of complex sub-systems (e.g., liquid wastemanagement system). For example, at a higher architecture level multipleslide stations may be configured for high throughput by virtue ofparallel processing. While the present system does require the addedsteps of applying and then peeling a membrane article from a slidesurface, the “cost” of these additional steps is more than offset byseveral gains including speed, reduction in fluid volume consumption,and reduction/elimination in fluid volume wastes.

The invention provides other advantages. For example, most or all of anentire tissue testing system could be integrated into a singleminiaturized station. For example, 7 reagents plus 2 rinses could bepackaged as separate wells on a single disposable “micro-fluidic card”to provide IHC staining of a single slide. A micro fluidic approachallows for the possibility of integrating individualized slide fluidicspermitting a different instrument design space. The entire station couldbe the size of, e.g., a small hand-held camera. Plumbing and large scalemechanics could be eliminated. Miniaturization and integration offersimplicity, design for quality control, robustness, and various types ofcost reductions. Modules may be ganged for higher throughput whileproviding true parallel processing means.

While the foregoing invention has been described largely in connectionwith aqueous-based fluids, the invention advantageously also may be usedwith non-aqueous fluids. In such case, there would be no need to employa hydrophobic element as the second substrate as described in theforegoing. Thus, many variations are possible which remain within theconcept and scope of the invention.

1. An apparatus for treating a biological sample with a liquid reagent,comprising first and second substrates having facing surfaces defining aspace therebetween in which said biological sample may be treated withsaid liquid reagent, wherein said first substrate comprises a relativelyrigid fluid impermeable element for supporting said biological samplewhile said second substrate comprises a relatively flexible gaspermeable element backed by a liquid impermeable element.
 2. Theapparatus of claim 1, wherein said first substrate comprises a glassslide.
 3. The apparatus of claim 2, wherein said biological specimencomprises a sectioned tissue sample carried on said slide.
 4. Theapparatus of claim 2, wherein said biological sample comprises acytological preparation carried on said slide.
 5. The apparatus of claim2, wherein said biological sample comprises a tissue sample arraycarried on said slide.
 6. The apparatus of claim 2, wherein saidbiological sample comprises a DNA microarray carried on said slide. 7.The apparatus of claim 2, wherein said biological sample comprises aprotein microarray carried on said slide.
 8. The apparatus of claim 1,wherein said second substrate comprises a gas permeable hydrophobicelement.
 9. The apparatus of claim 1, wherein said second substratecomprises a gas permeable hydrophobic porous flexible membrane.
 10. Theapparatus of claim 9, wherein said gas permeable hydrophobic porousflexible membrane comprises a polytetrafluoroethylene tape or sheet. 11.The apparatus of claim 9, wherein said gas permeable hydrophobic porousflexible membrane comprises a micro-porous semipermeable tape or sheet.12. The apparatus of claim 9, wherein said gas permeable hydrophobicporous flexible membrane is laminated to a liquid impermeable backingelement.
 13. The apparatus according to claim 1, wherein said liquidreagent comprises a biological stain or biological reagent.
 14. Theapparatus according to claim 1, wherein said liquid reagent comprises anaqueous based solution.
 15. The apparatus according to claim 1, whereinsaid liquid reagent is spread substantially uniformly between saidfacing surfaces.
 16. The apparatus according to claim 1, wherein saidliquid reagent is substantially free of gas bubbles.
 17. The apparatusaccording to claim 1, wherein said liquid reagent has a film thicknessof about 7-360 μm.
 18. The apparatus of claim 1, wherein said liquidreagent has a volume of about 100-300 microliters.
 19. The apparatus ofclaim 1, wherein said liquid reagent has a volume of about 5-50microliters.
 20. The apparatus of claim 1, wherein said liquid reagenthas a volume of about 20 microliters.